Simultaneous Ultra-Sensitive Detection of Structural and Single Nucleotide Variants Using Multiplex Droplet Digital PCR in Liquid Biopsies from Children with Medulloblastoma

Medulloblastoma is one of the most common types of brain tumors in children. During and after treatment with surgery, chemotherapy, and/or radiotherapy, children with this disease are monitored with imaging and cerebrospinal fluid analysis for the detection of tumor cells. These methods are not always sensitive or specific enough to confirm or rule out residual disease. Here, we develop a laboratory test based on the genetic makeup of medulloblastomas in 12 children. We analyze liquid biopsies (cerebrospinal fluid and blood plasma) for specific genetic fragments leaking from the individual tumors and find molecular traces of disease in 75% (9/12) of children overall. None of the children had malignant cells in the cerebrospinal fluid. We propose that this test could open up new technical possibilities to track measurable residual disease in children with medulloblastoma in order to further risk-adapt treatment, but first, larger studies of the approach at standardized time points are warranted.Medulloblastoma is a malignant embryonal tumor of the central nervous system (CNS) that mainly affects infants and children. Prognosis is highly variable, and molecular biomarkers for measurable residual disease (MRD) detection are lacking. Analysis of cell-free DNA (cfDNA) in cerebrospinal fluid (CSF) using broad genomic approaches, such as low-coverage whole-genome sequencing, has shown promising prognostic value. However, more sensitive methods are needed for MRD analysis. Here, we show the technical feasibility of capturing medulloblastoma-associated structural variants and point mutations simultaneously in cfDNA using multiplexed droplet digital PCR (ddPCR). Assay sensitivity was assessed with a dilution series of tumor in normal genomic DNA, and the limit of detection was below 100 pg of input DNA for all assays. False positive rates were zero for structural variant assays. Liquid biopsies (CSF and plasma, n = 47) were analyzed from 12 children with medulloblastoma, all with negative CSF cytology. MRD was detected in 75% (9/12) of patients overall. In CSF samples taken before or within 21 days of surgery, MRD was detected in 88% (7/8) of patients with localized disease and in one patient with the metastasized disease. Our results suggest that this approach could expand the utility of ddPCR and complement broader analyses of cfDNA for MRD detection.